Our work focuses on specialized synapses in the inner retina. We have expanded our study of inhibitory synaptic connections made by amacrine cells within the inner retina, to understand how feedforward and feedback inhibition contributes to signal processing in this network. We previously discovered that A17 amacrine cells provide rapid GABAergic feedback to rod bipolar cell ribbon synapses via a release process that is independent of membrane depolarization or voltage-gated calcium channels (Chavez, et al., 2006). This rapid feedback may be essential to prevent the rapid depletion of readily-releasable vesicles from the rod bipolar cell synaptic terminal (Singer and Diamond, 2006). Our most recent work indicates that reciprocal feedback from A17s extends the range over which these synapses encode luminance and compute contrast (Oesch and Diamond, submitted). Feedback from other amacrine cells weakly modulates the synaptic gain but does not change the operating range. Our understanding of ribbon synaptic physiology in bipolar cells is limited to rod bipolar cells. We don't know as much about synaptic transmission from cone bipolar cells, because it is very difficult to obtain synaptically coupled cone bipolar - ganglion cell pairs. We have crossed mouse lines with genetically encoded markers identifying specific types of bipolar and ganglion cells that are very likely to be connected. In particular, we have utilized mouse lines in which type two cone bipolar cells (CBC2) can be visualized. We find that CBC2s make reciprocal synaptic connections with AII amacrine cells. We are studying bidirectional communication between these two cell types, and we are also studying how the rod bipolar cell signal is shaped by the AII amacrine cell before it is passed to the CBC2. Our preliminary findings suggest that AIIs preferentially transmit information about contrast, but not luminance, to CBCs. In addition, AIIs pass signals with different temporal dynamics to ON and OFF cone bipolar cells, likely reflecting fundamental differences between electrical and chemical synapses. We are examining various cellular and synaptic processes that underlie these transformations, and a manuscript is in preparation. We have collaborated with Donald Zack (Johns Hopkins) to examine the physiological characteristics of retinal ganglion cells derived from stem cells (Sluch, et al., Sci. Rep. 5, 16595). We find that these cells develop diverse firing properties that are consistent with different ganglion cell subtypes at different stages of development. We also found that ESC-derived ganglion cells express functional glutamate receptors. A revised manuscript has been submitted for publication. We also have collaborated with Wai Wong (NEI) to examine roles for microglia in preserving circuit and synaptic morphology (Wang, et al., 2016 J. Neurosci.). In a mouse model of microglial depletion, many general features of retinal circuitry and photoreceptor morphology remained intact, yet bipolar cell responses (indicated by the electroretinogram b-wave) were impaired. Although photoreceptor synapses appeared normal at the light immunofluorescence level, we found using electron microscopy (EM) that photoreceptor synapses in microglia-depeleted retinas were dystrophic, with indistinct vesicles and irregular synaptic ribbons. Finally, we have extended our electron microscopy studies, in collaboration with Richard Leapman (NIBIB), to explore the detailed ultrastructure of synaptic ribbons in photoreceptors and rod bipolar cells. So far, EM tomography enables us to detect protein filaments that tether synaptic vesicles to the ribbon and the presynaptic membrane. We find that vesicles adopt different tethering relationships with respect to the ribbon and the presynaptic membrane, and we believe this may reflect morphological differences between docked and primed synaptic vesicles. We have obtained high-resolution three-dimensional images of rod bipolar cell ribbon synapses, using high-pressure freezing and EM tomographic techniques, under different physiological conditions that should give rise to different fractions of docked/primed vesicles. We are currently analyzing morphological differences between these different physiological states with the goal of determining the morphological substrate for vesicle priming at these synapses and to detect spatial patterns of exocytosis from the ribbon.